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Journal of Bacteriology, February 2008, p. 1298-1307, Vol. 190, No. 4
0021-9193/08/$08.00+0     doi:10.1128/JB.01639-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetics for Pseudoalteromonas Provides Tools To Manipulate Marine Bacterial Virus PM2

Hanna M. Kivelä,^1,2, Stefania Madonna,^3,, Mart Krupovì,² M. Luisa Tutino,^3,4 and Jaana K. H. Bamford^1*

Department of Biological and Environmental Science and Nanoscience Center, P.O. Box 35, FIN-40014, University of Jyväskylä, Jyväskylä, Finland,¹ Department of Biological and Environmental Sciences and Institute of Biotechnology, P.O. Box 56, FIN-00014, University of Helsinki, Helsinki, Finland,² Department of Organic Chemistry and Biochemistry, University of Naples Federico II, Monte Sant'Angelo, Via Cinthia, 4 80126, Naples, Italy,³ Faculty of Biotechnological Sciences, University of Naples Federico II, Naples, Italy⁴

Received 10 October 2007/ Accepted 1 December 2007

The genetic manipulation of marine double-stranded DNA (dsDNA) bacteriophage PM2 (Corticoviridae) has been limited so far. The isolation of an autonomously replicating DNA element of Pseudoalteromonas haloplanktis TAC125 and construction of a shuttle vector replicating in both Escherichia coli and Pseudoalteromonas enabled us to design a set of conjugative shuttle plasmids encoding tRNA suppressors for amber mutations. Using a host strain carrying a suppressor plasmid allows the introduction and analysis of nonsense mutations in PM2. Here, we describe the isolation and characterization of a suppressor-sensitive PM2 sus2 mutant deficient in the structural protein P10. To infect and replicate, PM2 delivers its 10-kbp genome across the cell envelopes of two gram-negative Pseudoalteromonas species. The events leading to the internalization of the circular supercoiled dsDNA are puzzling. In a poorly understood process that follows receptor recognition, the virion capsid disassembles and the internal membrane fuses with the host outer membrane. While beginning to unravel the mechanism of this process, we found that protein P10 plays an essential role in the host cell penetration.

Published ahead of print on 14 December 2007.

H.M.K. and S.M. contributed equally to this work.

Present address: Laboratory of Immunology and Allergology, Istituto Dermopatico dell'Immacolata (IDI)-IRCCS, Rome, Italy.